Leiomyomata (fibroids) are a non-malignant neoplasia of uterine smooth muscle cells (UtSMC) that afflict approximately 20% of all women and 80% of black women in the U.S. The most common tumor in women, fibroids cause severe pain and bleeding as well as infertility. Though temporary respite from the symptoms can be achieved by medical and surgical interventions, the only treatment that prevents recurrence is hysterectomy. More than 200,000 hysterectomies are performed annually in the U.S., with direct health care costs exceeding $2B; the emotional distress caused by loss of the uterus is incalculable. Despite its prevalence and severity, this condition has received sparse attention in the basic research community until quite recently. If we are to advance our understanding of the pathogenesis and improve treatment options for fibroids, it is critical to elucidate the mechanisms that underlie UtSMC proliferation. Work in our laboratory showed that the growth-arrest protein CCN5 was nearly absent in human fibroids, yet abundant in the normal myometrium of the same patients. We have also demonstrated that the CCN5 protein blocks human UtSMC proliferation in culture and in animal models by >80%. Gene profiling indicates that CCN5 regulates clusters of genes that control mitogenic signal transduction and cell cycle progression. These observations underpin our central hypothesis that CCN5 has a key role in maintaining normal smooth muscle function in the uterus, and that dysregulation of CCN5 expression is a major factor in the pathogenesis of fibroids. A corollary hypothesis is that administration of CCN5 will reduce or prevent fibroid formation. To test these hypotheses, in this revised proposal we will carry out structure-function analyses to determine the mechanisms by which CCN5 regulates UtSMC function using a combination of in vitro and in vivo model systems. Using immunocompromised mice implanted with human fibroid UtSMC engineered to express CCN5, we will determine if CCN5 or related peptides can suppress fibroid formation. These experiments are designed to provide a comprehensive assessment of the effects of CCN5 in human UtSMC from the molecular to the physiologic and pharmacologic levels. We hope the results of the proposed work will provide new insights into the pathophysiologic role of CCN5 in fibroids, and lead to novel therapeutic targets for treating this important women's health issue. PUBLIC HEALTH RELEVANCE: The goal of this project is to elucidate the mechanism of action of the protein CCN5 on uterine leiomyoma cells, and to determine if CCN5 has potential therapeutic value in the treatment of fibroids.